The use of DNA/RNA hybridization probes as basic research tools of molecular biology has flourished since publication of the seminal papers of Grunstein et al., Proc. Nat. Acad. Sci. USA 72:3961 (1975) and Southern, J. Mol. Biol. 98:503 (1975), which describe hybridization techniques employing radiolabeled polynucleotide probes.
The potential of polynucleotide probes as tools of clinical diagnosis is now apparent. To date, however, the outstanding majority of workers have employed probes comprising radioisotopically labeled atoms, which permit autoradiographic detection of probe/analyte hybrids with a reasonable degree of sensitivity. The applications of polynucleotide probes in clinical diagnosis have been highlighted by Lewin, Science, 221:1167 (1983), and Klausner et al., Bio/Technology, 1:471 (1983).
Conventional radiolabeled probes offer acceptable detectability thresholds, but can require lengthy periods for autoradiographic detection, and can introduce additional expenses associated with personnel monitoring and disposal of radioactive materials. Thus, nonradiometric polynucleotide probes with high sensitivity are of acute interest to the clinical and research communities as convenient, inexpensive and safe alternatives of isotopically-labeled probes.
Ward et al., published European Patent Application No. 82301804.9, publication No. 0663879, disclose probe compositions comprising purine, 7-deazapurine, or pyrimidine bases covalently coupled to a moiety capable of forming a detectable complex with a polypeptide. This moiety is coupled to purine bases at the 8-position, to deazapurine bases at the 7-position, and to pyrimidine bases at the 5-position. The resulting modified nucleotides are incorporated into DNA by nick-translation techniques.
Heller et al., published European Patent Application No. 82303701.5, publication No. 0070687, disclose a hybridization method for identification of target polynucleotides which employs light-labeled, single stranded polynucleotide reagents. Hybridization is detected by light emission, the intensity of which is related to the concentration of target polynucleotides in a sample mixture. The use of chemiluminescent, fluorescent, and phosphorescent reporter systems is disclosed. Heller et al., published European Patent Application No. 82303699.1, publication No. 0070685, disclose a homogeneous light-emitting hybridization assay employing luminescent-labeled first and second single-stranded reagent segments, which are hybridized in close proximity to one another along a target polynucleotide, such that nonradioactive energy transfer occurs between the light-emitting labels of the first segment and the fluorescent second segment. At least one of the light labels is of an absorber/emitter type, such that energy absorbed from the other light label is re-emitted at a secondary wavelength. Since such secondary emissions only occur if hybridization has taken place, the intensity of emission at the secondary wavelength is related to the concentration of target polynucleotide in the sample matrix.
These approaches to the design of efficient nonradiometric polynucleotide probes illustrate alternatives now being explored for application in novel diagnostic systems. It has now been found that ultrasensitive probe compositions can be prepared in the form of conjugates of polynucleotides and enzymatic zymogen activators, which serve to initiate cascade amplification detection rections.